This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mycobacterium tuberculosis is a highly successful human pathogen that has evolved to survive within the host. Our long-term goals are to delineate the mechanisms by which M. tuberculosis interferes with the microbicidal functions of macrophages and evades host immune responses. Understanding the molecular mechanisms underlying M. tuberculosis-host interactions is important for designing therapeutic interventions and vaccine strategies. We have identified Rv2224c, a predicted protease, as being critical for survival in macrophages and in vivo. We hypothesize that Rv2224c is an important virulence factor that proteolytically cleaves M. tuberculosis substrates and modulates the host immune response. While proteases are known to be key virulence factors in other bacterial pathogens, the function of proteases in M. tuberculosis pathogenesis has been largely unexplored. Our preliminary studies indicate that Rv2224c is exported to the cell envelope of mycobacteria. A mutant disrupted in the protease is impaired for growth in macrophages and in vivo. Mice infected with a protease mutant show markedly reduced lung pathology and survive longer than wildtype-infected mice and we have new evidence that Rv2224c modulates host innate immunity. In this project we are exploring the biochemical and molecular basis for protease function in M. tuberculosis, including characterizing a candidate substrate. We are also studying the role of protease in host-pathogen interactions. The specific aims are: 1. Biochemical characterization of Rv2224c and its potential substrate(s) in M. tuberculosis;2. Study the immune response induced by Rv2224c in vivo;3. Dissect the modulation of macrophage function by the protease. Characterizing how Rv2224c modulates immune responses will provide insights into understanding how M. tuberculosis evades host immunity, and help in designing vaccines to protect against tuberculosis disease.